Cellular oxidative metabolism leads to the generation of reactive oxygen species (ROS). Antioxidant mechanisms have evolved to regulate the steady-state flux of ROS and minimize their cytotoxicity. The antioxidant potential of cells is largely governed by both low-molecular-weight reductants and by antioxidant enzymes. While the intracellular determinants of ROS flux by vascular cells have been well characterized in recent years, the extracellular determinants of ROS flux and their regulation are less well understood. For the last two cycles of this award, we have studied a key antioxidant enzyme that regulates extracellular peroxide flux, glutathione peroxidase-3 (GPx-3). We studied the biochemistry and molecular cell biology of this selenoprotein in order to understand its role in regulating the bioavailability of endothelial nitric oxide and in modulating platelet activation in the vasculature. While GPx-3 can effectively reduce both hydrogen peroxide and lipid peroxides to water and lipid alcohols, respectively, recent kinetic studies suggest that hydrogen peroxide is the preferred substrate. Because extracellular hydrogen peroxide can affect endothelial cell-surface receptor signaling (at low concentrations) and extracellular oxidant stress (at high concentrations), the factors that govern its production, molecular actions, and metabolic fates in the vasculature warrant study. The central hypothesis of this proposal is that GPx-3 is a principal determinant of extracellular hydrogen peroxide flux from the endothelial cell, and a key regulator of the transmembrane cycling and actions of hydrogen peroxide in the endothelial cell. To test this hypothesis, we propose three specific aims. We will first assess the role of GPx-3 in eliminating hydrogen peroxide and will examine the reductive cofactor(s) required for its optimal activity in the extracellular microenvironment of the endothelial cell. Second, we will determine the role of hydrogen peroxide in regulating the expression of GPx-3 at the transcriptional, posttranscriptional, and translational levels. Third, we will examine the interactions between GPx-3 and its intracellular counterpart GPx-1 on the transcellular metabolism and cycling of hydrogen peroxide, and its consequences for "outside-in" and "inside-out" signaling in endothelial cells. These studies should provide insight into the complex relationships among hydrogen peroxide flux, extracellular antioxidant potential, and cell signaling and oxidant stress in the endothelial cell.